The present invention relates to plant genetic engineering and particularly to plastid transformation in higher plants. The invention provides novel promoter sequences useful for the expression of foreign genes of interest in various plant species.
Chloroplast genes are transcribed by an RNA polymerase containing plastid-encoded subunits homologous to the xcex1, xcex2 and xcex2 xe2x80x2 subunits of E. coli RNA polymerase. The promoters utilized by this enzyme are similar to E. coli "sgr"70-promoters consisting of xe2x88x9235 and xe2x88x9210 consensus elements (G. L. Igloi and H. Kossel, Crit. Rev. Plant Sci. 10, 525, 1992; W. Gruissem and J. C. Tonkyn, Crit. Rev. Plant. Sci. 12:-19, 1993) Promoter selection by the plastid-encoded RNA polymerase is dependent on nuclear-encoded sigma-like factors (Link et al. 1994, Plant promoters and transcription factors, Springer Verlag, Heidelberg, pp 63-83). In addition, transcription activity from some promoters is modulated by nuclear-encoded transcription factors interacting with elements upstream of the core promoter (L. A. Allison and P. Maliga, EMBO J., 14:3721-3730; R. Iratni, L. Baeza, A. Andreeva, R. Mache, S. Lerbs-Mache, Genes Dev. 8, 2928, 1994, Sun et al., Mol. Cell Biol. 9:5650-5659, 1989). These factors mediate nuclear control of plastid gene expression in response to developmental and environmental stimuli.
The existence of a second nuclear encoded polymerase transcription system in plastids has been demonstrated. However, the relevant nucleic acid sequences required for transcription initiation comprising the novel regulatory elements of this system have yet to be elucidated. It is an object of the present invention to provide these novel genetic elements. Incorporation of these regulatory elements into specific plastid directed DNA constructs enables greater flexibility and range in plant species available for plastid transformation, and facilitates ubiquitous expression of foreign proteins and/or RNAs and are useful in non-green plastids.
Promoters contain distinct DNA sequence information to facilitate recognition by the RNA polymerase and initiation of transcription leading to gene expression. In accordance with the present invention, promoters have been discovered which function in both monocots and dicots. These promoter elements may be used to advantage to express foreign genes of interest in a wider range of plant species. Additionally, the promoter elements of the invention drive expression of exogenous genes in non-green tissues. It is an object of the present invention to provide DNA constructs and methods for stably transforming plastids of multicellular plants containing such promoters. The DNA constructs of the invention extend the range of plant species that may be transformed.
The promoters recognized by plastid-encoded plastid RNA (PEP) polymerase have been well characterized in photosynthetic tissues such as leaf. The utility of PEP promoters for expression of foreign proteins in non-green tissues is demonstrated herein. The nuclear-encoded plastid (NEP) polymerase transcription system of the present invention directs expression of plastid genes also in roots, seeds, meristematic tissue and/or leaves. In most plants, including maize, cotton and wheat, plant regeneration is accomplished through somatic embryogenesis (i.e., involving meristematic tissue). In a preferred embodiment of the invention, efficient plastid transformation in these crops will be greatly facilitated, through the use of the NEP and PEP plastid transcription system and promoters of the present invention.
Particularly preferred promoters for use in the constructs of the invention are the clpP -111 (SEQ ID NOS: 15, 16, 30 and 31) promoters for the transformation of monocots and dicots and the pclp -53 promoter for transformation in dicots. Homologous clpP promoters from other plant species are contemplated to be within the scope of the present invention.
Other preferred promoters for use in expressing foreign genes of interest in the plant plastid in non-green tissues are PEP promoters present in the 16SrDNA operon, SEQ ID NOS: 28 and 29. Additional promoter elements suitable for use in the present invention are the rpoB and atpB promoters.
The NEP promoters of the invention are incorporated into currently available plastid transformation vectors such as those described in pending U.S. application Ser. No. 08/189,256, and also described by Svab and Maliga., Proc. Natl. Acad. Sci. USA, 90, 913 (1993). Protocols for using such vectors are described in U.S. Pat. No. 5,451,513. The disclosures of the three references cited above are all incorporated by reference herein. To obtain transgenic plants, plastids of non-photosynthetic tissues are transformed with selectable marker genes expressed from NEP promoters and transcribed by the nuclear-encoded polymerase. Likewise, to express foreign proteins of interest, expression cassettes are constructed for high level expression in non-photosynthetic tissue, using the NEP promoter transcribed by the nuclear-encoded plastid RNA polymerase. In another aspect of the invention, PEP promoters of the invention are incorporated into currently available plastid transformation vectors and protocols for use thereof.
In yet another aspect of the invention, the NEP transcription system also may be combined with the "sgr"70-type system through the use of dual NEP/PEP promoters. In transforming DNA constructs, the promoters are arrayed in tandem, operably linked to the coding region of the foreign gene of interest. As used herein, the term transcription unit refers to isolated DNA segments which comprise the essential coding regions of one or more exogenous protein(s) of interest. Such transcription units may also contain other cis elements for enhancing gene expression, such as enhancer elements. Transcription units are operably linked to the promoters of the invention, such that expression of the transcription unit is regulated by said promoter. Particularly preferred promoters for use in combination are the Prrn PEP promoters combined with the clpP type II NEP promoter in dicots and the Prrn PEP promoter combined with the clpP Type I NEP promoter for use in both monocots and dicots. A suitable Prrn promoter has the following sequence (SEQ ID NO: 32) 5xe2x80x2-GCTCCCCCGC CGTCGTTCAA TGAGAATGGA TAAGAGGCTC GTGGGATTGA CGTGAGGGGG CAGGGATGGC TATATTCTG GGAGCGAACT CCGGGCGAAT ACGAAGCGCt TGGATACAGT TGTAGGGAGG GATT-3xe2x80x2.
Homologous PEP and NEP promoters from a variety of plant species corresponding to those listed above are also considered to fall within the scope of the present invention. The transforming DNA also contains 3xe2x80x2 regulatory regions of plant or bacterial origin to effect efficient termination of transcription. An exemplary 3xe2x80x2 regulatory region is shown in FIG. 13, SEQ ID NO: 27.